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针对趋化因子受体CCR5的SPA-WGA药物筛选方法 被引量：1

Drug screening model targeted to CCR5 based on[^(35)S]GTPγS scintillation proximity assay
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摘要 目的建立针对趋化因子受体CCR5的药物筛选SPA-WGA法[35S]GTPγS结合实验方法。方法通过降低SPA-WGA小球用量和对其他各种因素进行优化,如细胞膜用量、GDP浓度、体系孵育时间和样品连续测量时间,建立了SPA-WGA法[35S]GTPγS结合实验方法。结果最后优化得到实验条件为:100μl反应体系,0.1 mg SPA-WGA小球,10μgCHO-CCR5细胞膜,10μmol.L-1GDP和0.3 nmo.lL-1[35S]GTPγS,室温孵育1.5 h并且在1 200 m in之内完成所有样品测量。该法与传统抽滤法比较,测量得到RANTES的EC50分别为:1.40 nmol.L-1和2.90 nmol.L-1,测量得到SCH-C的IC50分别为:2.95 nmo.lL-1和2.73 nmo.lL-1,测量结果相似,均呈现出较好的剂量效应关系。利用该法筛选54个化合物,筛到3个IC50在1~10 nmol.L-1的化合物。这3个化合物是否具有H IV-1进入抑制剂的潜在开发价值,还有待体外抗H IV-1实验的进一步验证和筛选。结论此方法操作简便,灵敏性和重现性好,且可高通量和自动化,该法适用于能与Gi家族蛋白偶联的GPCR的高通量药物筛选。 Aim To develop a high throughput screening model targeted to CCR5 based on [ 35^ S ] GTPγS scintillation proximity assay. Methods Membranes expressing hCCR5 were incubated for 60 min with GDP, [ 35^ S] GTPγS and SPA-WGA beads with agonist or antagonist at room temperature. Then the beads were centrifuged and radioactivity was measured with scintillation counter. The assay, which measures the level of G protein activation following RANTES occupation of CCR5, can be used to determine the degree of agonism and the potency of compounds acting at CCRS. Result A new in vitro assay was established for the high throughput screening of CCR5 after minimizing of the amount of SPA-WGA beads and optimizing of other factors including the amount of CCR5 membranes, the concentration of GDP, the incubation time course and the counting time course.The EC50 value of RANTES or the IC50 value of SCH-C gained by using [ 35^ S] GTPγS scintillation proximity assay is good and is similar to using [ 35^ S ] GTPγS filtration assay. Among 54 small molecular compounds screened by using [ 35^S] GTPγS scintillation proximity assay, 3 compounds with IC50 values among 1 - 10 nmol · L ^- 1 will be further testified on their anti-HIV activities by using HIV-1 Neutralization Assays. Conclusion The [ 35^ S ] GTPγS scintillation proximity assay is easily and safely operated, automatable, economical and repeatable. And the assay is feasible by high throughput screening for the receptors coupling to Gi protein such as CCR5
作者 陈仁海 吕燕慧 胡琼莹 裴钢 陈力
机构地区 中国科学院上海生命科学研究院生物化学与细胞生物学研究所
出处 《中国药理学通报》 CAS CSCD 北大核心 2006年第10期1266-1271,共6页 Chinese Pharmacological Bulletin
基金 国家"十五"重大科技专项资助项目(No2004AA2Z3443) 上海市科学技术委员会科技攻关计划资助项目(No03DZ19216)
关键词 趋化因子受体CCR5 亲和闪烁法 [35^S]GTPγS结合 实验方法 高通量筛选 chemokine receptor CCRS scintillation proximity assay （ SPA ） [ 35^ S ] GTPγS binding assay high throughput screening
分类号 R341.2 [医药卫生—基础医学] R392.11 [医药卫生—免疫学]
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中国药理学通报
2006年 第10期

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